Vectors, cells and processes for pyrimidine deoxyribonucleosides production

ABSTRACT

Novel DNA constructs and host cells comprising the same are disclosed. DNA constructs comprise a transcription unit (e.g. operon) comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin or a uridine kinase gene and/or a dCTP deaminase gene. In preferred embodiments the constructs comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin further comprise DNA sequences encoding for thymidylate synthase and/or transcription units comprising sequences encoding for uridine kinase preferably together with dCTP deaminase. In particularly preferred embodiments, the host cells comprise constructs having all of the above characteristics wherein the host cell displays repressed or no uracil DNA glycosylase activity. This may be achieved by removal of the host cell ung gene. Use of host cells in the manufacture of pyrimidine deoxyribonucleotides e.g. thymidine is also disclosed.

This application is filed pursuant to 35 U.S.C. 371 as a United StatesNational Phase Application of International Application No.PCT/GB00/02357 filed Jun. 30, 2000, which is a continuation in part ofU.S. Ser. No. 09/345,492 filed Jul. 1, 1999, now abandoned, which claimsbenefit of U.S. Ser. No. 60/141,827 filed Jul. 1, 1999.

FIELD OF THE INVENTION

The present invention relates to the production of pyrimidines, purinesand derivatives thereof e.g. deoxyribonucleosides, using geneticallymodified cells comprising novel DNA constructs.

BACKGROUND OF THE INVENTION

Thymidine is useful as a pharmaceutical intermediate, particularly forthe chemical synthesis of azidothymidine (“AZT,” sold under thetrademark ZIDOVUDINE). Although ZIDOVUDINE-type AZT was one of the firsttherapies developed for HIV/AIDS, it continues to have important andexpanded use (Langreth, R., The Wall Street Journal, Nov. 21, 1995, ppB12). ZIDOVUDINE-type AZT is valuable particularly when used incombination therapies such as a combination with lamivudine (also knownas 3TC), sold under the trademark EPIVIR. This lamvudine and 3TCcombination is sold under the trademark COMBIVIR. Although the HIV viruscan mutate to form resistance to either AZT or 3TC, COMBIVIR-typenucleotide-analog combination is particularly effective because thereverse transcriptase apparently cannot be resistant to both nucleosideanalogues at the same time (Larder, B. A. et al., Science 269: 696-699,1995). ZIDOVUDINE-type AZT is also useful in conjunction with HIVprotease inhibitor type drugs (Waldholz, M., The Wall Street Journal,Jan. 30, 1996, pp B1), and in the treatment of HIV infected pregnantwomen in order to reduce the frequency of infection of the fetus atbirth. In 1997 about 600,000 children died of AIDS contracted from theirmothers at birth. ZIDOVUDINE-type AZT taken for several months prior tobirth can reduce the transmission of the virus to infants by two-thirds.Thymidine produced by chemical synthesis used in the manufacture of AZTis a very significant cost.

In U.S. Pat. No. 5,213,972 (McCandliss & Anderson, hereinafter “the '972patent”), the entire contents of which are incorporated herein byreference and to which the reader is specifically referred, a processfor the production of pyrimidine deoxyribonucleoside (PdN) is disclosed(see in particular examples 7 to 14 of the '972 patent). A replicatablemicroorganism comprising and expressing a DNA sequence encoding apyrimidine deoxyribonucleotide phosphohydrolase that converts a PdNmonophosphate to a pyrimidine deoxyribonucleoside is taught. Moreparticularly, McCandliss & Anderson, supra, describe a fermentationmethod that can be used to produce thymidine that involves theexpression of deoxythymidylate phosphohydrolase (dTMPase) from theBacillus bacteriophage PBS1. This type of enzyme has been found innature expressed by bacteriophages that do not contain thymidine intheir DNA, but instead incorporates compounds like deoxyuridine orhydroxymethyl-deoxyuridine.

In the thymidine fermentation described in the '972 patent, the enzymesthat degrade thymidine (thymidine phosphorylase and uridinephosphorylase) have been removed by mutation so that thymidineaccumulates. Thus, the use of the dTMPase enzyme helps create thepathway to allow thymidine synthesis. An expression of dTMPase alone,however, may not assure a commercially viable level of thymidineproduction. Accordingly, there is a continuing need to enhance theproduction of thymidine by cells expressing dTMPase in order to makethymidine production by fermentation commercially viable, by loweringthe production cost relative to the current chemical synthesis methods.

The biochemical pathway for pyrimidine deoxynucleotide production, forexample, in E. coli is highly regulated at the levels of transcriptionand translation as well as at the protein level by mechanisms includingattenuation, feedback inhibition and enzyme activation. Neuhard, J. andR. A. Kelln, Biosynthesis and Conversion of Pyrimidines, Chapter 35 [In]Neidhardt, F. C. et al. [eds] “Escherichia coli and Salmonella Cellularand Molecular Biology”, Second Edition, Vol. I, pp580-599, ASM Press,Washington D.C., 1996. The expression of dTMPase and elimination ofthymidine breakdown by mutations in the deoA (thymidine phosphorylase),udp (uridine phosphorylase) and tdk (thymidine kinase) genes andtherefore resulting expression products results in thymidine synthesisin E. coli but not at a commerically viable level.

SUMMARY OF THE INVENTION

The biosynthesis of purines and pyrimidines involves a common step ofreducing a ribonucleoside diphosphate (in some species triphosphate) toits corresponding deoxy analog. In the overall process the reduction ofthe ribose moiety to 2-deoxyribose requires a pair of hydrogen atomswhich are ultimately donated by NADPH and H⁺. However, the immediateelectron donor is not NADPH but the reduced form of a heat stableprotein called thioredoxin or glutaredoxin and at least one otherunidentified source since the E. coli ribonucleotide reductase systemstill works in trxA (thioredoxin) grx (glutaredoxin) double mutants(Neuhard and Kelln, supra). The reducing equivalents of the reducedthioredoxin are transferred to ribonucleoside diphosphate reductasewhich carries out the reduction process. Manipulation of, for example,this step could prove useful in improving the commerical production ofpurine and pyrimidine deoxynucleosides.

It is an object of the present invention to provide novel DNA constructse.g. vectors and genetically modified microorganisms comprising saidvectors particularly for use in the production of recoverable amounts,especially commercially useful amounts, of pyrimidine and purinedeoxynucleosides.

It is also an object of the present invention to provide processes whichrepresent an improvement over McCandliss and Anderson described supra.

In accordance with one aspect of the present invention there is provideda DNA construct comprising a transcriptional unit which comprises aribonucleotide reductase gene and a thioredoxin gene or a uridine kinasegene and/or a dCTP deaminase gene.

In one embodiment the DNA construct comprises a transcriptional unitwhich comprises a ribonucleotide reductase gene and a thioredoxin gene.

In another embodiment the DNA construct comprises a transcriptional unitwhich comprises a uridine kinase gene and/or a dCTP deaminase gene.

Preferably the DNA construct comprises a transcriptional unit whichcomprises a uridine kinase gene and a dCTP deaminase gene.

Most preferably the DNA construct comprises a transcriptional unit whichcomprises a ribonucleotide reductase gene and a thioredoxin gene and auridine kinase gene and a dCTP deaminase gene.

In accordance with another aspect of the present invention there isprovided a modified host cell comprising a DNA construct according tothe invention.

In accordance with yet another aspect of the present invention there isprovided a culture medium comprising the modified host cells of theinvention and processes for the production of a purine or pyrimidine,for example thymidine comprising the use of said modified host cells.

In one embodiment the host cells comprise a DNA construct whichconstruct comprises a transcription DNA unit (e.g. operon) which unitcomprises DNA sequences encoding for ribonucleotide reductase andthioredoxin in which said reductase preferably displays less sensitivityto allosteric inhibition than a wild type host cell equivalent orcounterpart wherein said cell further comprises one or more of thefollowing features:

(a) a transcription unit (e.g. operon), preferably located on said DNAconstruct, comprising DNA sequences encoding for (and preferablyheterologous with respect to host cell equivalent) thymidylate synthase;

(b) a transcription unit (e.g. operon), preferably located on said DNAconstruct, comprising DNA sequences encoding for uridine kinase andpreferably dCTP deaminase; and

(c) repressed or absent Uracil DNA glycosylase activity.

In another embodiment the DNA construct for use in the production ofrecoverable amounts of pyrimidine and derivatives thereof, in particularpyrimidine deoxyribonucleosides such as thymidine, comprises atranscription unit (e.g. operon) which unit comprises (preferablyheterologous) DNA sequences encoding for uridine kinase and/or dCTPdeaminase.

Genetically modified host cells comprising and expressing the constructand culture medium comprising the modified host cells are also provided.

This aspect is based, in part, on the observation that host cellscomprising DNA encoding for uridine kinase and/or dCTP deaminase,optionally togather with additional genes as suggested in U.S. Pat. No.5,213,972 required for thymidine production, lead to a significantimprovement in thymidine production.

The respective aspects of the present invention disclose for the firsttime a plurality of advances on the teaching of U.S. Pat. No. 5,213,972to provide improved DNA constructs and host cells comprising theconstructs for use in the commercial production of pyrimidinedeoxyribonucleosides, particularly thymidine.

Other objects, features and advantages of the present invention willbecome apparent from the following description. It should be understood,however, that these represent preferred embodiments of the invention andare by way of illustration only. Various modifications and changeswithin the spirit and scope of the invention will become apparent tothose skilled in the art.

PREFERRED EMBODIMENTS OF THE INVENTION

The construct of the present invention may be chromosomal or morepreferably extra-chromosomal e.g. located on a vector.

Vectors of the present invention include plasmid, virus, transposons,minichromosome or phage, preferably plasmid. The vector comprising thetranscription unit may be introduced into the host cell according to anyconvenient method known to those skilled in the art, e.g. P1transduction, electroporation or transformation. Suitable host cellsuseful in the present invention include eukaryotes and prokaryotes (e.g.Bacterium). Prokaryotes include E. coli, Salmonella, Pseudomonas,Bacillus, strains and mutants thereof. E. coli is preferred due to thelarge amount of information, genetic tools and mutant alleles that areavailable. It is particularly preferred that a method of transduction isavailable for the host cell of choice to enable mutations to be readilymoved from one host cell to another and facilitate genetic mutation ofthe host without requiring direct mutation whenever a new mutation isdesired.

The present inventors have found that the use of bacteriophage T4 nrdA,nrdB and nrdC genes are particularly useful for encoding the reductaseand thioredoxin in E. coli. See Sjöberg, B. M. et al., EMBO J.,5:2031-2036 (1986); Tseng, M.-J., et al., J. Biol. Chem. 263:16242-16251(1988); and LeMaster, D. M., J. Virol. 59:759-760 (1986). Morespecifically, a very significant improvement in E. coli thymidineproduction was achieved through the cloning and expression of the T4bacteriophage nrdA and nrdB genes coding for ribonucleotide reductasetogether with T4 nrdC coding for thioredoxin since the T4 ribonucleotidereductase cannot use E. coli thioredoxin. The T4-coded ribonucleotidereductase was found to be relatively insensitive to control byallosteric inhibition in vitro compared to the E. coli enzyme (Berglund,O., J. Biol. Chem. 247:7276-7281, 1972). For example, unlike the E. colienzyme (Berglund, O., J. Biol. Chem. 247: 270-7275, 1972) the T4ribonucleotide reductase is not inhibited by dATP, but actuallystimulated by DATP and ATP (Berglund, O., J. Biol. Chem. 247:7276-7281,1972).

DNA sequences encoding for the ribonucleotide reductase (e.g. nrdA andnrdB genes) and thioredoxin (e.g. nrdC gene) are preferably heterologouswith respect to host cell DNA and preferably derived from T phage(preferably E. coli T bacteriophage), particularly T “even” phages e.g.T2, T4 or T6. See Campbell, A. M., Bacteriophages, Chapter 123, InNeidherdt, supra; and Mathews, C. K. et al. (eds.) Bacteriaophage T4,American Society of Microbiology, Washington, D.C., 1983. The term“derived from” is intended to define not only a source in the sense ofits physical origin but also to define material which has structuraland/or functional characteristics which correspond to materialoriginating from the reference source.

Another useful feature of the T even phage enzyme is its substratespecificity. The normal E. coli ribonucleotide reductase uses UDP as asubstrate only poorly since the K_(m) for UDP is about 10 fold higherfor UDP than CDP (Neuhard and Kelln, supra). However, the T4 enzyme hasonly a two-fold difference in K_(m) (Berglund, O., J. Biol. Chem. 247:7276-7281, 1972) between CDP and UDP substrates allowing two routes todUTP synthesis. Although there have been attempts to obtain functionalexpression of T4 ribonucleotide reductase in E. coli, previous effortswere only successful in expressing the components separately and coulddemonstrate activity only by mixing in vitro (Tseng, M.-J., P. He, J. M.Hilfinger, and G. R. Greenberg, J. Bacteriol. 172: 6323-6332, 1990).Whilst not being bound by theory, the inventors believe that perhaps dueto the lack of the usual pattern of feedback inhibition, expression ofT4 ribonucleotide reductase in E. coli is lethal and it must becarefully conditionally expressed. Further envisaged are genes thatencode precursor forms of the reductase and/or thioredoxin which areprocessed to produce a mature form. Such processing may proceed viavarious intermediate forms.

Vectors of the present invention preferably comprise a regulatoryelement (e.g. promoter such as lambda P_(L), operator, activator,repressor such as lambda repressor, particularly a temperature sensitivevariant, and/or enhancer), appropriate termination sequences initiationsequences and ribosome binding sites. The vector may further comprise aselectable marker. Alternatively, regulatory elements (particularlylambda repressor) may be located on the host cell chromosome. It ispreferred that nrdA and nrdB are arranged in the vector downstream (interms of reading frame) from nrdC. In particular, it is preferred thatnrdB is arranged downstream from nrdA. Thus a most preferred arrangementis a vector comprising an operon comprising nrdCAB.

The T4 ribonucleotide reductase is not devoid of feedback-control invivo (J. Ji, R. G. Sargent, and C. K. Mathews, J.Biol.Chem. 266:16289-16292, 1991; and Berglund supra). To promote ribonucleosidediphosphate reduction further e.g. for thymidine production, the genecoding for the regulatory subunit, nrdA, may be modified by, forexample, a mutational approach to create an enzyme capable of increasedthymidine production due to e.g. a reduced sensitivity to allostericinhibition for example inhibition by the enzyme's immediate product orinhibition by a product resulting from a downstream event.

In order to construct T4 nrdA mutants, site-directed mutagenesis may beused to modify or change (e.g. substitute) gene bases encoding aminoacids suspected to alter e.g. dTTP binding site involved in allostericregulation. Analysis of the amino acid sequence of T4 ribonucleotidereductase revealed a segment that appears to fit well with a postulatedconsensus sequence thought to be involved in dTTP binding (E. M.McIntosh and R. H. Haynes, Mol. Cell. Biol. 6:1711-1721, 1986). Severalchanges may be made in this region of the T4 ribonucleotide reductaseusing oligonucleotide-directed mutagenesis. The general approach may bemodelled after the effort of More et al. (More, J. T., J. M. Ciesla,L.-M. Changchien, G. F. Maley and F. Maley, Biochemistry 33: 2104-2112,1994) to reduce the dTTP binding of deoxycytidylate deaminase. Onemutation, ⁷⁹Ala to Ile, in the T4 nrdA appeared to be very useful. Forexample, the thymidine productivity of strains containing the ⁷⁹Ala tolie mutant in T4 nrdA evaluated by a shake flask fermentation method wassignificantly increased. As demonstrated below, the present inventorsachieved at least 25% increase over the parent strain without thissingle change.

Although the ⁷⁹Ala to Ile is one successful example, those skilled inthe art will now realize that many other amino acid changes to thisregion are now possible to obtain the desired effect, that being toputatively disrupt dTTP binding, but not disrupt the enzyme's basicfunctionality. For example, substitution of ⁷⁹Ala with other amino acidsdisplaying similar side chains to lie (e.g. leucine, valine) may beutilized. Modifications of position 79 in conjunction with othermodifications (e.g. mutations) within the postulated consensus regionare also envisaged. Deletion of one or more amino acid positions in theconsensus region and introduction of synthetic DNA into the region areother approaches available to those skilled in the art.

In another aspect of the present invention there is provided a host cellcomprising a construct which construct (e.g. vector) comprises atranscriptional unit comprising DNA sequences encoding for heterologousribonucleotide reductase and thioredoxin which reductase is lesssensitive to allosteric inhibition than the wild type host cellequivalent or counterpart. It will be apparent to those skilled in theart that determining the relative sensitivity of a candidateheterologous reductase to allosteric inhibition compared to the wildtype host cell equivalent is a matter of routine experimentation andobservation.

Transcription units comprising the DNA sequences e.g. nrdA, nrdB andnrdC genes are preferably operons wherein the nrd genes are arranged intandem. This permits transcription of these genes as a single mRNAtranscript. In order to minimize unproductive energy expenditure by thehost cell and further to minimize plasmid size, it is preferred that theoperon contains only genetic sequences required in the encoding ofreductase and thioredoxin (including any regulatory or controlelements). This may necessitate the removal of superfluous DNA (forexample, the unusual intron in the phage T4 nrdB gene, Sjoberg, B-M., etal EMBO J.5: 2031-2036, 1986).

In other preferred embodiments, vectors of the present invention for usein for example the production of thymidine further comprise DNAsequences encoding for thymidylate synthase (e.g. the td gene). See e.g.Chu, F. K. et al., Proc. Natl. Acad. Sci. USA 81:3049-3053 (1984); Chu,F. K. et al., J. Bacteriol. 169:4368-4375 (1987). The purpose of usingthis enzyme is to improve control over the levels of deoxyuridineproduced and in particular the relative impurity level of deoxyuridinerelative to thymidine. The dTMPase enzyme is not completely specific fordTMP. With a higher K_(m) than for dTMP, the PBS1 dTMPase will alsoutilize dUMP as substrate to produce deoxyuridine (Price, A. R., Methodsin Enzymol. 51: 285-290, 1978). Deoxyuridine creates a significantproblem for thymidine purification. Therefore, one way to reducedeoxyuridine production is to efficiently convert dUMP to dTMP byincreasing the level or effectiveness of thymidylate synthase such thatthe internal concentration of dUMP always remains very low.

The thymidylate synthase gene (td) may be heterologous with respect tothe host cell and it is preferred that td is derived from (in the sensedefined supra) T bacteriophage, e.g. T “even” phage and in particular T4phage td. Although td may be located in its own transcription unit, itis preferred that td is located in the same transcription unit e.g.operon as nrd genes. Moreover, it is preferred that td is located in thesame operon downstream (in terms of reading frame) from the nrd genes.

McCandliss and Anderson, supra, amplified the E. coli thymidylatesynthase gene in plasmids pCG138 and pCG148 (see Table 5, of the '972patent) and it was found to be partially effective in reducingdeoxyuridine. The T4 thymidylate synthase is much more effective whichis surprising in light of the fact that the E. coli enzyme is notthought to be controlled by any type of allosteric regulation (Neuhardand Kelln, supra). The E. coli enzyme K_(m) for dUMP, 4 μM (Wahba, A. J.and M. Friedkin, J. Biol. Chem. 237: 3794-3801), and the T4 enzyme K_(m)for dUMP, 2.73 μM (Maley, F., L. LaPat-Polasko, V. Frasca and G. F.Maley, Functional domains In T4 Thymidylate Synthase as probed bysite-directed mutagenesis, Chapter 29 [In] Karam, J. D. [ed] “MolecularBiology of Bacteriophage T4”, American society for Microbiology,Washington, D.C., 1994, pp 322-325), are similar and cannot explain thelarge difference in effectiveness. Whilst not being bound by theory, theinventors believe that the E. coli thyA has an internal transcriptiontermination sequence derived from an upstream gene that could beeffecting the expression level in plasmid clones (Bell-Penderson, D, J.L. Galloway Salvo, and M. Belfort, J. Bacteriol. 173:1193-1200, 1991).

In other preferred embodiments, host cells of the present invention,particularly for use in the commercial production of pyrimidinedeoxyribonucleosides e.g. thymidine comprise a transcription unit (e.g.operon) which unit comprises DNA sequences e.g. udk gene encoding foruridine kinase and preferably DNA sequences e.g. dcd gene encoding fordCTP deaminase. See e.g. Wang, L. and B. Weiss, J. Bacteriol.174:5647-5653 (1992); and Neuhard, J. and L. Tarpø, J. Bacteriol. 175:5742-5743.

The construct of this aspect of the invention may additionally comprisea transcription unit encoding for ribonucleotide reductase (nrdA andnrdB) and the thioredoxin (nrdC), or precursor forms thereof which arepreferably heterologous with respect to host cell DNA and preferablyderived from E. coli bacteriophage, particularly T “even” phages e.g.T2,T4 or T6.

Uridine kinase produces UMP and CMP from uridine and cytidine using GTP(or dGTP) as the phosphate donor. The reaction is inhibited by UTP andCTP (J. Neuhard and R. D. Kelln, Biosynthesis and Conversions ofPyrimidines, Chapter 35 [in] F. C. Neidhart et al. [ed], “EscherichiaColi and Salmonella Cellular and Molecular Biology”, Second Edition, ASMpress, Washington D.C.). The present inventors have found that the useof uridine kinase particularly together with dCTP deaminase leads to amarked improvement in the production of thymidine by host cellsincorporating these changes together with the teachings of '972 outlinedsupra. This observation is quite unexpected since uridine kinase, on thebasis of current information, has no direct role in pyrimidine de novobiosynthesis, moreover that its use would be beneficial in commercialprocesses for the production of pyrimidine deoxyribonucleosides. It ispreferred that udk and dcd genes are arranged in tandem in the sameoperon. Further envisaged are genes that encode precursor forms of theudk and dcd gene which are processed to produce a mature form. Suchprocessing may proceed via various intermediate forms. The udk and dcdgenes may be introduced into the construct (e.g. vector) from anysuitable source by methods well known to those skilled in the art forexample P1 transduction, electroporation or transformation.

The enzyme uracil DNA glycosylase, encoded by the ung gene, isresponsible for degrading DNA that has uracil incorporated in place ofthymine. Where host cells of the present invention are used in thecommercial production of e.g. thymidine, the internal cellularconcentration of dTTP may be lowered as a result of the utilization ofdTMP (a precursor of dTTP) in the production of thymidine. Accordingly,the present inventors have recognized that there Is potentially agreater propensity for uracil incorporation into the host DNA which maybe lethal to a wild type host due to the uracil DNA glycosylase activitycausing too many single stranded breaks in the host cell DNA. Thus, hostcells useful In the present invention may further display repressed(compared to the unmodified cell) or no uracil DNA glycosylase activity.This repression or absence may be achieved through various ways apparentto those skilled in the art. For example, antagonism (either total orpartial) of the ung gene expression products is one such approach byintroducing an antagonist of the functional enzyme (or precursorthereof) into the host cell. Other approaches include manipulating unggene expression by e.g. modifying regulatory elements of ung geneexpression or introducing mutations into the ung gene itself such thatung gene product expression displays little or no uracil DNA glycosylaseprotein and/or activity. Another approach is to delete the ung gene (orfunctionally critical parts thereof) from host cell DNA. The absence orlow level of uracil DNA glycosylase activity may be a feature of thehost cell without the need for further manipulation.

In preferred embodiments of the present invention, each of the advancestaught herein are incorporated into a host cell. The nrd, td, udk anddcd genes may be located on separate constructs but it is preferred thatthey are all located on the same construct e.g. vector. Thus in aparticularly preferred embodiment of the present invention, a modifiedhost cell is provided in which the cell comprises a DNA construct (e.g.vector) comprising a transcription DNA unit (e.g. operon) which unitcomprises DNA sequences encoding for preferably a T even phage, e.g. T4)a modified ribonucleotide reductase and thioredoxin in which saidreductase preferably displays less sensitivity to allosteric inhibitionthan wild type host cell equivalent or counterpart wherein saidconstruct further comprises:

(a) a transcription unit (e.g. operon) encoding for (preferablyheterologous with respect to host cell equivalent or counterpart)thymidylate synthase and;

(b) a transcription unit (e.g. operon), encoding for uridine kinase andpreferably dCTP deaminase; and in which the host cell displays repressedor absent uracil DNA glycosylase activity.

Host cells modified according to the present invention are particularlyuseful in the commercial production of pyrimidine deoxynucleosides. In aparticularly advantageous use of the present invention, E. coli hostcells comprising (harboring) a plasmid modified according to the presentinvention (particularly in conjunction with the teachings of the '972patent) may be used in the commercial production of thymidine. Thus,host cells modified according to the present invention may furthercomprise dTMPase derived from e.g. PBS1 and the mutations taught in the'972 patent, e.g. deoA, tdk-1 and udp-1.

Generally, a fermentation method is employed which involves submergingthe cells in a culture medium contained within a suitable vessel.Following culturing under appropriate conditions, produced thymidine isharvested and purified (enriched), if necessary, to pharmaceutical gradeaccording to standard protocols. The purified thymidine may then be usedin the production of medicaments, e.g. pharmaceutical compositions suchas AZT.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example only and withreference to the following figures in which:

FIG. 1 illustrates, schematically, a route for the construction ofpCG366. It should be noted that the plasmids are not drawn to scale.

FIG. 2 illustrates, schematically, a route for the construction ofpCG374 and pCG375.

FIG. 3 illustrates a map for plasmid pCG532.

FIG. 4 illustrates growth and thymidine production by recombinant E.coli strain CMG2451 (Ung⁺) and CMG2492(Ung⁻) hosting a plasmid pCG366(nrdCAB td) according to example 10.

FIG. 5 illustrates thymidine production in a 30-liter fermentor by E.coli CMG2451 (pCG532).

FIG. 6 illustrates TdR and UdR (mg/L) obtained according to thepurification protocol of example 12.

EXAMPLE 1 Cloning of T4 nrdCAB Genes and Demonstration of Activity

The bacteriophage T4 nrdAB genes were cloned by performing thepolymerase chain reaction (PCR) with isolated phage T4 DNA. The primersfor PCR nrdA gene were: 5′-TAT TCT AGA CGA TTT TCA AGT TGA GGA CTT ATGC-3′ (Seq. id 1); and 5′-TAT ATC GAT AAT TCA TTA CAA TTT ACA CGC TGCAC-3′ (Seq. id 2).

The restriction site XbaI was the introduced at the beginning of thenrdA in the amplified DNA, and ClaI was introduced at the 3′ end ofnrdA. The primers for PCR amplification of nrdB gene were:

5′-TAT ATC GAT AAA TGT AAA TTT AAG GAT TCT AAA TG-3′ (Seq. id 3) and

5′-TAT GTC GAC TCC TTA AAA GTA TTT TTT AAA ACT C-3′ (Seq. id 4).

The restriction site ClaI was thus introduced at the beginning of thenrdB, and ApaI was introduced at the end of nrdB in the amplified DNA.The PCR fragments were cloned into plasmid vectors as illustrated inFIG. 1 according to techniques known to those skilled in the art. Thecloned nrdAB genes were confirmed by enzyme activity assay. The T4 nrdCgene was cloned into pKC30 producing plasmid pDL51 (LeMaster, D. M., J.Virology 59: 759-760, 1986 ) and was supplied by D. LeMaster (Dept ofBiochem., Univ. Wisconsin, Madison, Wis). The gene was sub-cloned into aplasmid with nrdAB genes as illustrated in the FIG. 1. The sources ofstarting materials and background information used in FIG. 1 are listedin Table 1.

A synthetic transcriptional terminator was used for the construction ofpCG198 and pCG301 (see FIG. 1 and Table 1). Specifically, plasmid pBCsk⁺ obtained from Stratagene (La Jolla, Calif.) was digested withrestriction enzyme ApaI and Asp718 I. Synthetic DNA containing theECOTGP transcription termination sequence (d' Aubenton Carafa et al., J.Mol. Biol. 216: 839-843, 1990) was then ligated replacing the originalsequence

TABLE 1 Genealogy of Plasmid pCG366 and pCG532 and Materials Source Pro-Vector Plasmid Gene(s) moter origin Marker Derivation PACYC177 amp kanp15A amp kan Chang and Cohen (1978) J. Bacteriol. 134:1141-1156. ATCC37031 pBC sk+ Cam Plac ColE1 Cam Stratagene, 11011 North Torrey PinesRoad, La Jolla, CA 92037. pBluescript Amp Plac colE1 Amp Stratagene,11011 North Torrey II ks Pines Road, La Jolla, CA 92037. ATCC 87047pBR322 amp tet colE1 amp tet Bolivar et al. (1977) Gene 2:95-113. pDL51T4 nrdC colE1 Amp LeMaster, J. Virol. 59: 759-760, 1986 pKC30 Amp λP_(L)colE1 Amp Shimatake, and Rosenberg, (1981) Nature 292:128. ATCC 37286pKTdΔl T4 td colE1 Amp West et al. (1986) J. B. C. 261:13446-13450.pT7/T3/18 Amp phage colE1 Amp Life Technologies, INC.,9800 T7/T3 MedicalCenter Dr., Rockville, MD 20897. pTZ18U Amp colE1 Amp Bio-Rad, 2000Alfred Nobel Dr., Hercules, CA 94547. pUC18 Amp Plac colE1 AmpYanisch-Perron et al. (1985) Gene 33:103-119. ATCC 37253 pUC19 Amp PlaccolE1 Amp Yanisch-Perron et al. (1985) Gene 33:103-119. ATCC 37254pCG173 λP_(L) vector λP_(L) colE1 Amp λP_(L) in pBluescript II ks pCG196T4 nrdC colE1 Amp T4 nrdC in pUC19 pCG198 Cam Plac colE1 Cam Synthetictranscription terminator in pBC sk+ pCG301 λP_(L) vector λP_(L) colE1Cam λP_(L) in pCG198 pCG308 T4 nrdC λP_(L) colE1 Cam T4 nrdC in pCG301,LeMaster, J. Virol. 59: 759-760. 1986 pCG312 T4 nrdA PT7 colE1 Amp T4nrdA in pT7/T3/18 pCG318 NrdB PT7 colE1 T4 nrdB in in pBluscript II kspCG323 NrdAC λP_(L) colE1 Cam T4 nrdAC in pCG301 pCG326 NrdABC λP_(L)colE1 Cam T4 nrdABC in pCG301 pCG332 NrdAB λP_(L) colE1 Cam T4 nrdAB inpCG301 pCG334 NrdAB λP_(L) colE1 Cam T4 nrdAB in pCG301 pCG337 NrdCABλP_(L) colE1 Cam T4 nrdCAB in pCG301 pCG343 NrdCAB λP_(L) colE1 Cam T4nrdCAB in pCG301 pCG356 Td λP_(L) colE1 Cam T4 td in pCG301 pCG358 dcd,udk colE1 Amp E. coli dcd, udk in pUC18 (pUC18) pCG360 NrdCAB, td λP_(L)colE1 Cam T4 nrdCAB td in pCG301 pCG366 NrdCAB, td λP_(L) colE1 Cam TetT4 nrdCAB, td in pBR322, Amp^(S) (pBR322) pCG374 dcd, udk p15A amp E.coli dcd, udk in pACYC177 pCG376 λP_(L) , vector λP_(L) p15A amppACYC177 with λP_(L) & MCS of pCG301 pCG464 T4 nrdA — (pTZ18U) AmpXbal/HindIII fragment with nrdA Xbal/HindIII colE1 sequence cloned intoXbal/HindIII sites fragment of pTZ18U (BioRad Laboratories) pCG492 T4nrdA Xbal/ — (pTZ18U) Amp PCG464 with mutation in T3 nrdA HindIII colE1sequence causing ⁷⁹ala→ile alteration Fragment pCG494 NrdCAB td λP_(L)colE1 Cam KpnI/AflII fragment from pCG494 (pBR322) Tet inserted intopCG366 adding ⁷⁹ala→ ile mutation to nrdA pCG532 NrdCAB, td, λP_(L)colE1 Cam udk,dcd cloned into pCG494 udk, Dcd (pBR322)

between ApaI and Asp718 I endonuclease sites. This fragment recreatedthe ApaI recognition sequence, but destroyed the Asp 718 I recognitionsequence in the new plasmid. The inserted DNA had the followingcomposition

5′-CGAGC CCGCCTAATG AGCGGGCTTT TTT TT-3′ 3′-CCGGGCTCG GGCGGATTACTCGCCCGAAA AAAAACATG-5′ (shown as respective strands In seq. id 5 and 6)produced from two oligonucleotides.

Plasmid pCG198 was combined with pCG173 that contains the lambda P_(L)promoter from plasmid pKC30 cloned into pBluescript II ks (Stratagene,La Jolla, Calif.) as shown in FIG. 1. Both pCB198 and pCG173 weredigested with Hind III and Asn I then ligated to create new plasmidpCG301 containing the lambda P_(L) promoter, multiple restriction enzymecloning sites, and followed by the ECOTGP terminator sequence copiedfrom the tryptophan operon leader peptide region.

T4 ribonucleotide reductase activity was measured by HPLC by a methodthat does not involve the use of radioisotopes and UDP substrate. Thedirect ribonucleotide reductase assay contained 1 mM NADPH, 1 mM DTT,0.5 mM dATP, 0.6 mM UDP, 20 mM Tris (pH 8.0) and 5 mM MgCl₂. Under theseconditions the E. coli ribonucleotide reductase is inhibited and is notdetected. The enzyme reaction (100 μL) was stopped by the addition of 10μL of 50% trichloroacetic acid (TCA). After 10 minutes on ice, thesamples are centrifuged in a microcentrifuge. The supernatant wasextracted 4 times with diethylether to remove the TCA. Five ml of Trisbuffer (1.0 M pH 8.0) was added followed by 2 μL of 40 mg/mL rattlesnake venom (Sigma), and the sample was incubated at 37° C. for 60minutes. The samples were then heated for 3 minutes at 70° C. followedby centrifugation for 5 minutes to remove precipitate. The volumes areequalized, then analysed by HPLC with an UV detector and deoxyuridine asthe standard. The column is a Spherisorb ODS-2, 5 micron, 250 mm×4.6 mmusing a 12 mM ammonium phosphate (pH 5.0) mobile phase and a flow rateof approximately 1.0 mL/minute. Results are shown in Table 2 for cellscontaining plasmid pCG343 demonstrating functional expression of T4ribonucleotide reductase.

TABLE 2 Ribonucleotide Reductase Activity Specific Acitivity StrainInduction condition nmol/10 min./mg protein CMG1093 Uninduced 0 CMG1093Induced 0 CMG1093/pCG343 Uninduced 0 CMG1093/pCG343 Induced 704.7

EXAMPLE 2 Derivation of Host Strain CMG2451 from Strain CMG1115

Strain CMG1115 is fully described in McCandliss & Anderson (U.S. Pat.No. 5,213,972). CMG1115 was the starting point for development describedherein. Strain CMG1115 was improved for thymidine productivity byselection for growth on medium containing 30 mg/L of 5-fluorouridinethat yielded strain CMG2401. Strain CMG2401 was then selected for growthon medium containing 30 mg/l of 3′-azido-3′-deoxythymidine which yieldedstrain CMG2404. CMG2404 requires L-proline for growth due to theinherited mutation Δ(lac-pro) from its original parent JM101. Hfr matingbetween CMG2404 and a Hfr stain CAG5053 (Singer, M. et al.Microbiological Review; 5:1-24, 1989) was performed according totechniques known to those skilled in the art and yielded strain CMG2434which is Lac⁺, Pro⁺. The udp (uridine phosphorylase) mutation in CMG2434still had partial uridine phosphorylase activity that was evident basedon thymine accumulation after induction of thymidine production. The udpmutation was reintroduced from CGSC5128 (E. coli Genetic Stock Center,Yale University) by phage P1 transduction according to techniques knownto those skilled in the art. The metE3079::Tn 10 from strain CAG18491was first transduced into CMG2434 to serve as a positive selectionmarker for transduction of udp. Then the udp-1 was transduced into themetE3079::Tn10 derivative of CMG2434 by selecting for growth withoutL-methionine methionine in the defined medium. The udp-1 derivative wasnamed strain CMG2451. The genealogy of CMG2451 is summarized in Table 3.

TABLE 3 Genealogy of E. coli Host Strain CMG2451 Strain GenotypeDerivation^(a) CMG1115 CMG1106 Tn5::dTMPase Insertion from (Tn5::dTMPasekan^(R)) pCG132. CMG2401 CMG1115 FUdR^(R) 5-fluoro-2′deoxyuridineresistance. CMG2404 CMG2401 AZT^(R) 3′-Azido-3′deoxythymidineresistance. CMG2434 CMG2404 Lac⁺Pro⁺ Repair Δ(lac-proAB) by conjugationwith CAG5053^(b). CMG2448 CMG2434 metE3079::Tn10 from CAG18491^(b).metE3079::Tn10 CMG2451 CMG2448 udp-1 udp-1 from CGSC5128^(c) andreplaced metE⁺tet^(a) metE3079::Tn10. ^(a)Mutations were at timesintroduced into E. coli strains by phage P1 transduction. If themutation has a selective marker, direct P1 transduction was used. If themutation has no selective marker, P1 cotransduction with the nearby Tn10insertion was used. ^(b)Singer, M., et al. Microbiological Review:53:1-24, 1989. ^(c)All CGSC strains can be obtained for E. coli GeneticStock Center, Yale University, P.O. Box 208104, New Haven, CT06520-8104.

EXAMPLE 3 Cloning and Expression of T4 td Gene into Thymidine ProductionPlasmid

The T4 td gene was cloned into pKTdΔl by West et al. (J. Biol. Chem261:13446-13450, 1986) without the 1017-base pair intron. The td genewas sub-cloned into pCG301 using two oligonucleotides as linkers withthe following sequences: 5′-GAT CCG GAG GAT AAA TGA AAC AAT ACC MG AAGATT TAA T-3′ (seq. id 7) and; 5′-TAA ATC TTG GTA TTG TTT CAT TTA TCC TCCG-3′ (seq. id 8).

The result plasmid was pCG356. The td gene from pCG356 was thensub-cloned into pCG343 to create pCG360 (FIG. 1). The tetracyclineresistance gene and plasmid replication origin from pBR322 (Bolivar, F.et al., Gene 2:95-113, 1977) was sub-cloned into pCG360 and formedpCG366. The thymidylate synthase activity was measured byspectrophotometric method of Wahba and Friedkin (J. Biol.Chem. 236:PC11-PC12, 1961). The results are shown in Table 4.

TABLE 4 Thymidylate Synthase Activity Specific Activity Strain ΔOD340/mgprotein/min CMG1093 −0.72 CMG1093/pKTdDl 3.24 CMG1093/pCG356 3.45CMG1093/pCG360 1.43

EXAMPLE 4 Shake Flask Data Showing the Value of T4 td Gene on ThymidineProduction and Deoxyuridine Reduction

The shake flask fermentation was used to evaluate the thymidineproductivity of different E. coli recombinants. The shake flaskfermentation broth and methods used here, and in other examples, isdescribed in Example 6 below. In this case 20 mL volume per flask wasinoculated with 2 ml of seed culture and incubated in a 30° C. shaker.At about 10 OD 600 nm, the flasks were transferred into a 37° C. shakerfor 30 minutes to mildly induce the λP_(L) promoter. Then the flaskswere transferred into a 35° C. shaker to continue the fermentation.Glucose was fed during fermentation as needed, and pH was adjusted toabout 7 with ammonia according to the color of phenol red. The thymidineconcentration was measured by HPLC using a Sperisorb ODS-2, 5 micron,250-mm×4.6 mm column, and a 25 mM ammonium phosphate (pH 3.3) mobilephase with a flow rate of approximately 1.5 ml/minute.

The strain CMG2451/pCG366 (T4 nrdCAB, td) was compared withCMG2451/pCG343 (T4 nrdCAB) in shake flask fermentation. Table 5 showsthat deoxyuridine concentration was reduced, and converted to thymidinein strain CMG2451/pCG366 due to the T4 td gene.

TABLE 5 Thymidine Production-Effect of Thymidylate Synthase at 66 hours.Thymidine Stain (mg/l) Deoxyuridine (mg/l) % Deoxyuridine CMG2451/pCG3431198 1728 144.2 CMG2451/pCG366 3327 206 6.2

EXAMPLE 5 Construction of the ⁷⁹Ala to Ile T4 Ribonucleotide ReductaseMutant

The mutation was introduced by using site-directed mutagenesis based ona method described by Kunkel (Kunkel, T. A., Proc. Natl. Acad. Sci. USA,82: 488-492, 1985). All materials for mutant construction includingpTZ18U phagemid DNA, M13KO7 helper phage, bacterial strains E. coliCJ236 and MV1190, T7 DNA polymerase and T4 DNA ligase were provided inthe Muta-Gene in vitro mutagenesis kit from Bio-Rad Laboratories(Hercules, Calif.). At first, the XbaI/HindIII DNA fragment containingthe nrdA gene of T4 bacteriophage was isolated from plasmid pCG312(ChemGen Corp., Table 1 above). The XbaI/HindIII DNA fragment was clonedinto the XbaI/HindIII sites of the pTZ18U phagemid vector using standardprotocols (Sambrook, J., Fritsch, E. F. and Maniatis, T., MolecularCloning, Cold Spring Harbor Laboratory, New York ,1989). Theinsert-carrying phagemid pCG464 was introduced into E. coli CJ236. Thisstrain is deficient for dUTPase (dut) and uracil-N-glycosylase (ung)which results in an occasional substitution of uracil for thymine innewly synthesized DNA. Single stranded DNA of pCG464 containing uracilwas isolated from CJ236 according to Bio-Rad Laboratories InstructionalManual. This DNA (0.2 pMole) was annealed with 6 pMole of phosphorylatedprimer 5′-AGC AAA CAT TAA ACA GCG TGC AATTAC ATA TTG ATA ATC AGG TTC-3′(sequence id 9) containing the sequence of the desired mutation(underlined) coding for Ile instead of original ⁷⁹Ala.

Complementary strand DNA was synthesized by using T7 DNA polymerase asdescribed in the Bio-Rad protocol. The reaction products weretransformed into E. coli MV1190 containing a wild typeuracil-N-glycosylase, which degrades the uracil-containing parentalstrand, thus enriching for the mutant strand. Direct DNA sequencingusing the Silver Sequence DNA Sequencing System from Promega Corp.(Madison, Wis.) identified plasmids containing the desired mutation. Itappeared that all four analysed transformants contained plasmids withthe mutation. One of them was designated pCG492 and used for furtherexperiments. To check if the mutation affects thymidine synthesis it wasintroduced into production plasmid pCG366 (ChemGen Corp., Table 1 andFIG. 1). For this, the KpnI/AflII DNA fragment of pCG366 containing5′-part of the nrdA gene was replaced with KpnI/AflII fragment frompCG492 that contains the mutation. The new plasmid pCG494 was introducedinto production strain CMG2451 (ChemGen Corp., Example 2 and Table 4above). The effect of T4 nrdA mutation was evaluated by comparison ofthymidine production by CMG2451 (pCG494) and CMG2451 (pCG366) as shownin Example 6 below.

EXAMPLE 6 Shake Flask Fermentation for Thymidine Production usingCMG2451 (pCG366) and CMG2451 (pCG494)

The 250-mL baffle flasks containing 25 mL of production medium wereinoculated with 2 mL of a freshly grown seed culture in LB broth withappropriate antibiotic added. The cultures were grown in a 30° C. shakerat 250 rpm. When the OD₆₀₀ reached about 5, the flasks were transferredinto a 37° C. shaker for 30 min. Then the flasks were transferred into a35° C. shaker to continue the fermentation. The production medium hasthe following composition (g/L): Ardamine YEP-S (Red Star Yeast &Products, Milwaukee, Wis.)—10; CaCO₃—10; MgSO₄—0.4; phenol red—0.24;PP90BT (DMV International, Fraser, N.Y.)—4.5; sorbitol—20;chloramphenicol—0.03; trace elements (1000X)—1 mL/L. The trace elements(1000X) formulation is the following (g/L): boric acid—0.05; calciumchloride—20; cobalt sulfate—0.05; copper sulfate—0.01; ferroussulfate—20; ferric chloride—20; manganese sulfate—0.5; sodiummolybdenate—0.1; and zinc sulfate—0.1. At the time of induction 10 gramsper liter of Ardamine YEP-S was added. Glucose was fed during thefermentation on an average of every two hours (2.5 g/L). The pH in theflasks was maintained at approximately 7.0 through the addition of 4NNH₄OH as judged by the color of the phenol red indicator dye. The OD₆₀₀was read after sample dilution 1:10 into 10 mM H₂SO₄ to dissolve saltsin the medium. Thymidine concentration was measured by reverse phaseC-18 HPLC with an Alltech Sperisorb ODS-2 column and Shimadzuspectrophotometric detector at 260 nm. The mobile phase was a 25 mMNH₄H₂PO₄ (pH 3.3) in water at the constant rate of 1.5 mL/min.

The results of thymidine production by CMG2451 (pCG366) and CMG2451(pCG494) after 2, 17 and 25 hours after induction are presented in Table6. There were two repeats of the flasks in this experiment andvariability between duplicate flasks did not exceed 15%. The resultsshow that the T4 nrdA mutant performed better than the wild type nrdAstrain. This was confirmed in several independent shake flaskexperiments with the same bacterial strains.

TABLE 6 Thymidine production by CMG2451 (pCG366) and CMG2451 (pCG494)Specific activity (mg/L/OD) Strain 2 hours 17 hours 25 hours CMG2451(pCG366) 6.1 32.5 43.1 CMG2452 (pCG494) 8.5 36.1 51.4

EXAMPLE 7 Shake Flask Fermentation to Demonstrate the Effect of the E.coli udk Gene on Thymidine Production

The dcd gene or dcd udk operon were cloned into the pACYC177 vector.This vector with the p15a origin of replication is different from colE1based plasmids such as pCG366 and thus is compatible and can bemaintained in the same host with colE1 based plasmids. The details ofthe plasmid constructions resulting in pCG374 (udk dcd) or pCG375 (dcd)are shown in FIG. 2. The genes on these plasmids are expressed from thenative E. coli promoter of the udk dcd operon.

Using selection for ampicillin resistance, plasmids pCG374 and pCG375were introduced into CMG2451 (pCG366) to test the effect on thymidineproduction in the shake flask fermentation method described in Example6. The results at several time points are shown in Table 7. Although thespecific activity (thymidine per OD of cells) is similar both with andwithout the udk gene, the cells with the udk gene on the second plasmidpCG374 grew to a higher cell density and produced significantly morethymidine (5.8 g/L compared to 3.0 g/L for the strain with the dcd onlysecond plasmid). This result was not anticipated, as it is not clear whyuridine phosphorylase could have this effect.

Based on this data and other information, the udk gene was chosen forintroduction along with the E. coli dcd gene in the construction ofplasmid pCG532 (see Example 8 and FIG. 3).

TABLE 7 Comparison of the effect of second plasmids with dcd or dcd udkon thymidine production in the CMG2451 (pCG366) background. TimeThymidine Specific Thymidine (hour) O.D.600 (mg/l) (mg/l/OD) Base CMGCMG CMG2451 CMG2451 CMG2451 CMG Strain 241 2451 (pCG366) (pCG366)(pCG36G) 2451 And (pCG (pCG (pCG Plasmid 366) 366) 366) Second PCGpCG375 pCG374 pCG375 pCG374 PCG pACYC 374 with with with with 375 177with dcd dcd udk dcd dcd udk with based dcd dcd plasmid udk  8 hr 29.11229.94 889 884 30.5 29.5 18 hr 40.206 32.946 1721 1608 42.8 48.8 42 hr48.348 33.96 4020 2740 83.1 80.7 66 hr 57.498 28.614 5804 3012 100.9105.3

EXAMPLE 8 Cloning of the E. coli udk dcd Operon into Production PlasmidpCG494

The udk and the dcd genes of E. coli encode pyrimidine ribonucleotidekinase and dCTP deaminase, respectively. Both genes were mapped to a 3.4kb BamHI/PstI DNA fragment of lambda phage 355 of the Kohara genomiclibrary (Kohara, Y., Akiyama, K. and Isono, K., Cell 50, 495-508, 1987).It appears that udk is located upstream of dcd and transcribed in thesame direction as dcd (Neuhard, J. and Tarpo, L., J. Bacteriol. 175:5742-5743, 1993). The genes were cloned into production plasmid by twosteps.

At first, a 3.4 kb BamHI/PstI DNA fragment from lambda 355 was clonedinto the multiple cloning site of plasmid pUC18 (Yamisch-Perron, C.,Vieira, J. and Messing, J., Gene 33: 103-109, 1985) (plasmid pCG358).Then, the fragment was excised from the polylinker region of pCG358 withBamHI and SphI and cloned in place of a 0.7 kb Bg/II/SphI fragment(containing a portion of the tetracycline resistance gene) of productionplasmid pCG494. The final 14.7 kb plasmid pCG532 contains colE1compatibility group origin of replication, the chloramphenicolresistance gene, the udk and the dcd genes and the T4 bacteriophagenrdCAB (encode thioredoxin and two subunits of ribonucleotide reductase,respectively) and the T4 td (encodes thymidylate synthase) genes undercontrol of the P_(L) promoter of bacteriophage lambda. The T4 nrdA geneof pCG532 was previously changed by site-directed mutagenesis (⁷⁹Ala toIle).

A synthetic transcriptional terminator is located downstream of the tdgene to prevent transcriptional readthrough into the replication region.The genetic map of plasmid pCG532 is illustrated in FIG. 3.

EXAMPLE 9 Shake Flask Fermentation of Thymidine by CMG2451 (pCG494) andCMG2451 (pCG532)

Plasmid pCG532 containing the udk and the dcd genes of E. coli wasintroduced into production strain CMG2451. New strain CMG2451 (pCG532)was tested together with parent strain CMG2451 (pCG494) in shake flaskexperiments to compare thymidine production. The results of twoindependent experiments are shown in Table 8. The first experiment wasperformed as described above and samples were taken at 17 hours afterinduction. In the second experiment cells were induced at higher OD(about 9) and samples were taken at 3 hours after induction foranalysis. In both cases the strain containing the cloned udk and dcdgenes performed better than the parent strain.

TABLE 8 Thymidine fermentation by thymidin by CMG2451 (pCG494) andCMG2451 (pCG532) Experiment 1 Specific Activity Strain O.D 600 TdR(mg/L) (mg/L/OD) CMG2451 (pCG494) 16.5 599 36.3 CMG2451 (pCG532) 17.5867 49.5 Experiment 2 Strain CMG2451 (pCG494) 8.5 149 17.5 CMG2451(pCG532) 8.4 192 22.8

EXAMPLE 10 Addition of ung Mutation and its Effect on ThymidineProduction in Shake Flask Fermentation

An uracil DNA glycosylase negative strain was constructed by introducingan ung::Tn10 (Varshney, U., et al., J. Biol. Chem. 263:7776-7784, 1988)mutation into host CMG2451 using P1 transduction as described above, andwas named CMG2492. Plasmid pCG366 was introduced into CMG2492. Acomparison experiment between Ung and Ung⁺ strain for thymidinesynthesis in shake flasks is shown in FIG. 4 using the flask methoddescribed in Example 6. Cells were grown in 250 ml flasks at 30° C., andthymidine synthesis was induced by shifting temperature to 37° C. for 30min, then,. shifting to 35° C. The Ung host without uracil DNAglycosylase kept growing longer and made 30% more thymidine.

EXAMPLE 11 Thymidine Production in a 30 Liter Fermentor with StrainCMG2451 (pCG532)

The following conditions were used to produce thymidine in a 30-Literfermentor (B. Braun Biotec Biostat C) with strain CMG2451/532. The seedculture (500 mL)was grown in a 4 Liter baffle shake flask in LB medium(5 g/L Difco yeast extract, 10 g/L Difco tryptone, 5 g/L NaCl) with 30mg/L chloramphenicol and 25 mg/L kanamycin at 30° C. until 2.37 OD 600nm was reached with a final pH of 6.69.

The initial batch in the fermentor (12 Liters) containing thecomposition listed in Table 9 was sterilized at 121° C. for 55 minutes.After cooling a separately autoclaved solution (500 mL) was added toadjust the batch to 20 g/L sorbitol and 3.0 g/L MgSO₄7 H₂O. Also addedbefore inoculation was a sterile filtered solution (200 mL) designed toto adjust the initial batch to 30 mg/L chloramphenicol, 25 mg/Lkanamycin, 1 mg/L d-biotin, 10 mg/L thiamine and 10 mg/L nicotinic acid.

Three feed solutions were prepared: a) Cerelose 2001 (dextrosemonohydrate) 562 g/L with 2 mg/L biotin, 20 mg/L thiamine, 20 mg/Lnicotinic acid, and 30 mg/L chloramphenicol; b) sorbitol 717 g/L withwith 4 mg/L biotin, 40 mg/L thiamine, 40 mg/L nicotinic acid, and 60mg/L chloramphenicol; and c) crude nitrogen mixture containing 360 g/LAmberex 695 AG (Red Star Yeast & Products, Milwaukee, Wis.), 6 g/L PP90M(DMV International, Fraser, N.Y.) with 1X trace elements and 0.1 mL/LMazu DF10PMOD11 (BASF). The feed solutions were sterilized for 40 to 50minutes under 18 PSI steam pressure.

TABLE 9 Initial batch composition in 30 L fermentor ConcentrationComponent (g/L or mL/L) Sodium hexametaphosphate 4 KH₂PO₄ 4 (NH₄)₂HPO₄ 4CaCl₂ 0.4 Citric acid 0.5 Amberex 695 (Red Star Yeast & Products, 20Milwaukee, WI) PP90M (DMV International, Fraser, NY) 15 Tryptone (Difco,Detroit, MI) 5 1000X Trace elements (see Example 7)   1 mL/L CaCO₃ 5Glycine 1.83 Mazu DF10PMOD11 Defoamer (BASF 0.2 mL/L SpecialityProducts, Gurnee, IL)

The operating conditions were as follows: initial temperature 31° C.;RPM 600; air flow 3 LPM; pH 6.8; pressure 0 Bar. The dissolved oxygenwas controlled at 25% saturation using air flow rate control loop. TheRPM was increased to 750 RPM at 8 hours, 850 RPM at 9 hours and 950 RPMat the time of temperature shift from 31 to 35.5° C. at 9.2 hours whenthe culture reached 37.8 OD. Beginning at 9.7 hours back pressure wasapplied up to a maximum of 0.6 Bar to aid in oxygen transfer into theculture. The rate of temperature shift for induction of thymidinesynthesis was 0.2° C. per minute.

After the cell mass reached 20 OD at 600 nm (read after dilution in 50mM H₂SO₄to dissolve salts), 85 mL batch feeds of the sorbitol feedsolution (b) were made for each 5 OD increase in cell mass. At 16.7hours sorbitol batch feeding was stopped and dextrose monohydrate(glucose) feed (a) was started under the control of the DO PID controlloop of the B. Braun fermentor with a set point of 25%. Simply stated,when dissolved oxygen was below 25% sugar feed was off, and when DO wasgreater than 25% the sugar feed was set to on. The protocol selfregulates the glucose concentration keeping the concentration low, butdoes not allow the culture to starve for glucose for a very prolongedlength of time. Crude nitrogen feeds (500 mL) were made at 11 hours,16.2 hours, 21.2 hours, 28.2 hours, 33.2 hours and 40.5 hours.

The cell mass, thymidine and deoxyuridine accumulation during thefermentation are shown in FIG. 5.

EXAMPLE 12 Purification of Thymidine from Fermentation Broth

Dowex Optiptore-L-285 (The Dow Chemical Company) was suspended indeionized water and packed into a 48 mM diameter glass column making abed volume of about 500 mL. The column was washed with 500 mL of 5%NaOH, then washed with deionized water to until the effluent was pH 7.0.

500 mL Fermentation broth with thymidine (TdR) concentration of 4.890g/L and deoxyuridine (UdR) concentration of 1.040 g/L was loaded ontothe column. The column was washed with two bed volumes of deionizedwater. TdR and UdR were eluted by two bed volumes of 5% reagent alcohol(Ethanol 90.5%, Methanol 4.5%, Isopropyl alcohol 5%) followed by two bedvolumes of 10% reagent alcohol and two bed volumes of 15% reagentalcohol. The TdR and UdR in the 25 mL fractions is shown in FIG. 6. Thecolumn was regenerated by washing with 5% NaOH, then with deionizedwater to pH7.0 and the procedure was repeated. The fractions 91-160 werepooled together from the two separate runs and dried using a rotaryvacuum evaporator. Thymidine was re-dissolved in a minimal amount of hotwater and crystallized at 4° C. Then the crystals were re-crystallizedtwo times and dried in a 55° C. oven for 15 hours. A total of 979.8 mgof crystalline thymidine was obtained with a purity of greater than 99%that should be suitable for use as a pharmaceutical intermediate.

The side fractions containing both deoxyuridine and thymidine werepooled with mother liquors and reduced by a rotary vacuum evaporator toa final volume of 1200 mL with reduced alcohol. The total amount of TdRin this 1200 mL solution was 3571 mg. The total recovery (including the3571 mg of TdR side fractions) was 93.1%.

9 1 34 DNA Artificial Sequence Description of Artificial Sequence Primer1 tattctagac gattttcaag ttgaggactt atgc 34 2 35 DNA Artificial SequenceDescription of Artificial Sequence Primer 2 tatatcgata attcattacaatttacacgc tgcac 35 3 35 DNA Artificial Sequence Description ofArtificial Sequence Primer 3 tatatcgata aatgtaaatt taaggattct aaatg 35 434 DNA Artificial Sequence Description of Artificial Sequence Primer 4tatgtcgact ccttaaaagt attttttaaa actc 34 5 30 DNA Artificial SequenceDescription of Artificial Sequence DNA insert 5 cgagcccgcc taatgagcgggctttttttt 30 6 38 DNA Artificial Sequence Description of ArtificialSequence DNA insert 6 gtacaaaaaa aagcccgctc attaggcggg ctcgggcc 38 7 37DNA Artificial Sequence Description of Artificial SequenceOligonucleotide 7 gatccggagg ataaatgaaa caataccaag atttaat 37 8 31 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotide 8taaatcttgg tattgtttca tttatcctcc g 31 9 45 DNA Artificial SequenceDescription of Artificial Sequence Primer 9 agcaaacatt aaacagcgtgcaattacata ttgataatca ggttc 45

What is claimed is:
 1. A DNA construct comprising a transcriptional unitwhich comprises a ribonucleotide reductase gene wherein theribonucleotide reductase gene comprises a T4 nrdA gene modified tocomprise SEQ ID No.
 9. 2. A DNA construct as claimed in claim 1comprising a transcriptional unit further comprising a T4 nrdB gene anda thioredoxin gene wherein the thioredoxin gene is a T4 nrdC gene.
 3. ADNA construct according to claim 2 wherein the construct is a vector. 4.A DNA construct according to claim 3 wherein the vector is a virus,transposon, minichromosome or phage.
 5. A DNA construct according toclaim 3 wherein the vector is a plasmid, and wherein the modified T4nrdA gene the T4 nrdB gene, and the T4 nrdC gene are arranged in anoperon.
 6. A DNA construct according to claim 5 wherein the modified T4nrdA and T4 nrdB genes are located downstream of the T4 nrdC gene.
 7. ADNA construct according to claim 6 wherein the T4 nrdC gene is upstreamof the modified T4 nrdA gene and the modified T4 nrdA gene is upstreamof the T4 nrdB gene.
 8. A DNA construct according to claim 4, furthercomprising a regulatory element.
 9. A DNA construct according to claim8, wherein the regulatory element is selected from the group consistingof a promoter, an operator, a termination sequence, an initiationsequence and a ribosome binding site.
 10. A DNA construct according toclaim 9 wherein the promoter is the lambda P_(L) promoter or aderivative therefrom.
 11. A DNA construct according to claim 9 whereinthe termination sequence is a heterologous terminator sequence.
 12. ADNA construct according to claim 1 wherein the modified T4 nrdA gene ismodified such that the ribonucleotide reductase encoded by the unit isless sensitive to allosteric inhibition than the wild type equivalent ofsaid ribonucleotide reductase.
 13. A DNA construct according to claim 5wherein the construct further comprises a T4 td (thylidymate synthase)gene.
 14. A DNA construct according to claim 13 wherein the td gene islocated in the same operon as the nrdA, nrdB and nrdC genes.
 15. A DNAconstruct according to claim 14 wherein the td gene is locateddownstream from the modified nrdA, nrdB, and nrdC genes.
 16. A DNAconstruct according to claim 2 further comprising an E. coli uridinekinase gene or an E. coli dCTP deaminase gene.
 17. A DNA constructaccording to claim 16, wherein the DNA construct comprises both an E.coli uridine kinase gene and an E. coli dCTP deaminase gene.
 18. A DNAconstruct according to claim 17 wherein the E. coli uridine kinase geneis a udk gene.
 19. A DNA construct according to claim 17 wherein E. colidCTP deaminase gene.
 20. A modified E. coli host cell comprising a DNAconstruct according to any one of claims 1, 2, 3-6, 7-12, 13, 14, 15, or16-19.
 21. A modified E. coli host cell according to claim 20 whereinthe host cell displays repressed or no uracil DNA glycosylase activity.22. A modified E. coli host cell according to claim 21 wherein one ormore host cell DNA polynucleotides encoding for uracil DNA glycosylaseactivity have been modified so as to encode expression productsdisplaying repressed, low levels of, or no uracil DNA glycosylaseactivity.
 23. A modified E. coli host cell according to claim 22 whereinthe modified host cell DNA polynucleotide comprises an ung gene.
 24. Amodified E. coli host cell according to claim 21 wherein one or morehost cell DNA polynucleotides encoding for uracil DNA glycosylaseactivity have been removed.
 25. A modified E. coli host cell comprisinga DNA construct, which construct comprises a transcription unit, whichunit comprises a modified T4 nrdA gene comprising SEQ ID No 9, T4 nrdBgene and a T4 nrdC gene, wherein the modified T4 nrdA gene encodes aribonucleotide reductase which is less-sensitive to allostericinhibition than the wild-type equivalent of the reductase, and whereinsaid host cell further comprises one or more of the following: (a) atranscription unit located on said DNA construct, comprising athymidylate synthase gene heterologous to the thymidylate synthase goneof the host cell; (b) a transcription unit located on said DNAconstruct, comprising an E. coli uridine kinase gene; (c) atranscription unit located on said DNA construct, comprising an E. colidCTP deaminase gene; and (d) repressed or absent uracil DNA glycosylaseactivity.
 26. A modified E. coli host cell according to claim 25,wherein the ribonucleotide reductase gene is modified at a dTTP bindingsite.
 27. A modified E. coli host cell according to claim 25, whereinthe DNA construct comprises both the uridine kinase gene and the dCTPdeaminase gene.
 28. A modified E. coli host cell according to claim 25,wherein the cell comprises each one of the features of (a) to (d).
 29. Aprocess for the production of pyrimidine deoxyribonucleosides comprisingculturing a host cell according to claim
 20. 30. A process according toclaim 29 wherein the deoxyribonucleoside is thymidine.
 31. A culturemedium comprising a host cell according claim 20.